FLUOROMETRIC MEASUREMENT OF DEOXYRIBONUCLEIC ACID IN BONE MARROW CELLS THE MEASUREMENT OF MEGAKARYOCYTE DEOXYRIBONUCLEIC ACID

Abstract

The use of fluorochromes in the Feulgen reaction for the quantitative measurement of deoxyribonucleic acid (DNA) has been explored previously but is rarely applied. The occurrence of nonspecific fluorescence due to compounds other than DNA and the fading of the fluorochrome on excitation can cause variation in the measurements of nuclear fluorescence. The method of Ruch (in Introduction to Quantitative Cytochemistry, Wied, G. L., ed., Academic Press, 1966, p. 281) using auramine O as the fluorochrome has been modified to achieve improved nuclear fluorescence with a more linear rate of fading. A simple Zeiss microscope photometer has been used. Problems of instrumentation and optics, including lens changes during measurements on cells of widely varying sizes such as megakaryocytes, have been analyzed and correction factors have been calculated. Microfluorometric measurements of nuclear DNA are of the same order of accuracy as those made by integrating microdensitometry, without the need in the latter technique to compress large cells to a uniform thickness. Microfluorometry has been applied with the new staining method to analyze the ploidy distribution of bone marrow megakaryocytes.